Novel porous polymer monoliths adapted for sample preparation

ABSTRACT

A porous polymer monolith comprises a polymer body having macroporous through-pores that facilitate fluid flow through the body and an array of mesopores adapted to bind from the fluid flow molecules of a predetermined range of sizes, wherein the surface area of the monolith is predominantly provided by the mesopores. Also disclosed is a method of making a porous polymer monolith. The method includes forming a polymer body by phase separation out of a solution containing at least a monomer, a crosslinker and a primary porogen, whereby the body contains multiple macroporous through-pores, wherein the solution further contains a secondary porogen comprising oligomers inert with respect to the monomer and cross-linker but chemically compatible with the monomer so as to form mesostructures within the polymer body during said phase separation, and washing the mesostructures from the body to provide an array of mesopores such that the surface area of the monolith is predominantly provided by the mesopores.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/779,241, filed May 25, 2018 which is a U.S. National StageApplication of PCT/AU2016/051163, filed on 28 Nov. 2016, which claimspriority from Australian patent application no. 2015904917, filed on 27Nov. 2015, the entire disclosures of which is are incorporated herein byreference. To the extent appropriate, a claim of priority is made toeach of the above disclosed applications.

FIELD OF THE INVENTION

This invention relates generally to porous polymer monoliths, tosampling devices incorporating such monoliths, and to methods for theirpreparation.

BACKGROUND OF THE INVENTION

Porous polymer monoliths are a class of material having particularutility in sample preparation techniques in analytical chemistry. Theconventional method of synthesis involves forming a solution by mixingone or more monomers, a crosslinker, one or more porogens, a mediatorand an initiator. A commonly employed combination has styrene as amonomer, divinylbenzene (DVB) as the crosslinker, toluene as themediator and dodecanol as the porogen. This formulation is disclosed forexample, in the context of the formation of macroporous polymer beads inChing Wang et al, Journal of Polymer Science: Part A: Polymer Chemistry32, 2577-2588 (1994). With appropriate conditions, the mixture resultsin a monolith with macro through-pores and micropores with a pore sizebelow 20 Å.

For the majority of analytes these polymer monolith materials areeffectively non-porous with resulting low capacity. The principalapplications for the materials involve separations of macro-moleculessuch as proteins. A number of attempts have been made to increase thespecific surface area, including a hyper-crosslinking technique thatinvolves filling the through-pores with an open mesh of crosslinkedpolymers. These methods have tended to undo some of the advantages themonolithic material had in the first place.

International patent publications WO 2011/082449 and WO 2013/006904disclose the use of porous polymer matrix or monolith materials as mediafor the storage of biological fluids, including body fluids such aswhole blood or blood plasma. As described therein, porous polymermonoliths are highly crosslinked structures that can function as astationary support and consist of a fused array of micro globulesseparated by pores. In embodiments of particular interest, such porouspolymer monoliths are formed from one or more functionalised monomersincluding a hydrophilic monomer such as 2-hydroxyethylmethacrylate(HEMA) and a mixture of porogens including one or more alcohols and oneor more alkanes.

United States Patent Application Publication 20150211967 discloses asampling device, such as a pipette tip or a cartridge adapted for solidphase extraction (SPE), in which the flow path includes a bed of aporous polymer monolith selected to adsorb bioparticles from a matrixdrawn or dispensed through an inlet opening and the bed.

In a recent paper by Saba et al (‘Hierarchically Porous PolymerMonoliths by Combining Controlled Macro—and MicroPhase Separation’), J.Am. Chem. Soc. 2015, 137, 8896-8899, there is described a tunablenanoporous polymer monolith based on controlled polymerisation ofstyrene and DVB from a poly(lactide) macro-chain transfer agent in thepresence of non-reactive polyethylene oxide (PEO). Morphologies can betailored from mesoporous, with control over mean pore size, tohierarchically meso and macro porous. The paper describes the presenceof residual PEO in the cross-linked polystyrene matrix, said to improvethe wettability of the monolith and thereby creating a hydrophilicrather than hydrophobic structure. Moreover, it is said that the‘structure contains isolated macropores that are accessible through apercolating mesoporous network’. This structure will be a significantinhibiter to through-flow across the monolith. It is further evidentthat the abovementioned tunability inherently entails an effect on themacroporous structure when varying the mean mesopore size.

Accordingly, while the Saba et al paper does propose a technique forintroducing a mesoporous structure into a porous polymer monolith, theapproach here taken has a number of disadvantages and does notsatisfactorily overcome the problem of providing mesopores to improvebinding capacities while preserving macroporous through-flow. It is anobject of the invention to address this problem in an effective manner.

Reference to any prior art in the specification is not an acknowledgmentor suggestion that this prior art forms part of the common generalknowledge in any jurisdiction or that this prior art could reasonably beexpected to be understood, regarded as relevant, and/or combined withother pieces of prior art by a skilled person in the art.

SUMMARY OF THE INVENTION

The invention entails the realisation that the introduction of asuitably compatible secondary porogen into the synthesis of porouspolymer monoliths can result in the formation of an array of mesoporessubstantially independently of the macroporous structure. Moreover, itis possible to adapt the method to pre-select the mesopore size andthereby to achieve a tuneable analyte size cut off.

The invention accordingly provides, in a first aspect, a porous polymermonolith comprising a polymer body having macroporous through-pores thatfacilitate fluid flow through the body and an array of mesopores adaptedto bind from the fluid flow molecules of a predetermined range of sizes,wherein the surface area of the monolith is predominantly provided bythe mesopores.

By “predominantly” in this context is meant greater than 50%, preferablygreater than 65%, more preferably at least 85%, most preferably at least90%.

In a second aspect, the invention provides a method of making a porouspolymer monolith comprising:

-   -   forming a polymer body by phase separation out of a solution        containing at least a monomer, a crosslinker and a primary        porogen, whereby the body contains multiple macroporous        through-pores, wherein the solution further contains a secondary        porogen comprising oligomers inert with respect to the monomer        and cross-linker but chemically compatible with the monomer so        as to form mesostructures within the polymer body during said        phase separation, and    -   washing the mesostructures from the body to provide an array of        mesopores such that the surface area of the monolith is        predominantly provided by the mesopores.

By “mesopores” in the present context is meant pores of a size in therange 2-50 nm (20-500 Å). By analogy, references here to micropores areto pores of a pore size below 20 Å, and references to macropores are toa pore size greater than 500 Å.

In embodiments of the invention, the preferred mesopore size range is40-120 Å, more preferably 50-100 Å.

The invention further provides a porous polymer monolith made by themethod of the second aspect of the invention. This monolith may also bein accordance with the first aspect of the invention.

EMBODIMENTS OF THE INVENTION

In an embodiment of interest, the monomer is styrene, the crosslinkermay be Divinylbenzene (DVB), and the primary porogen may be dodecanol.In this embodiment, the secondary porogen comprises low molecular weightpolystyrene, ie. styrene oligomers, preferably of a molecule weight notgreater than 5000, more preferably not greater than 2000. Above amolecule weight of 5000, the oligomers become less soluble in thereaction mixture. Toluene is the preferred mediator and the initiatormay be azobisisobutyronitrile (AIBN) or benzoylperoxide (BPO).

Preferably, the styrene oligomers are structured, for example by lackingresidual double bonds, to minimise or prevent their participation in theprimary phase separation polymerisation reaction that forms the monolithbody. For example, styrene oligomers obtained by cationic polymerisationdo not contain any residual double bonds.

A preferred porous polystyrene monolith may have a bimodal pore sizedistribution with large, eg. 1 to 5, preferably 2 to 3 micron, macrothrough-pores and mesopores around 50-100 Å pore size. In the preferredmethod of the invention, these macro through-pores result from the phaseseparation polymerisation reaction between the styrene and thedodecanol, while these mesopores are the voids left behind following thewashing from the polymer body of the secondary porogen mesostructuresarising from the styrene oligomers. The washing effects dissolution andphysical washing away of the secondary porogen mesostructures.

In an alternate embodiment, derived from the porous polymer monolithsdisclosed in international patent publication WO 2011/082449, in whichthe monomer is a mix of methacrylates, and the primary porogens may be amix of at least one alcohol and at least one alkane, a suitablesecondary porogen would be a HEMA oligomer, which can e.g. besynthesised by reversible addition-fragmentation chain transfer (RAFT)polymerization. The principle is applicable to any monolithic recipewhere the secondary porogen is an oligomeric form of the monolithicpolymer.

Preferably, there is no or substantially no residual secondary porogenremaining in the monolith structure.

Preferably, the macro through-pores are substantially unmodified andunaffected by the addition of the secondary porogen during the formationof the polymer body.

Preferably, the mesopores contain sites, preferably hydrophobic, adaptedto bind molecules of predetermined character, structure, chemistry orsize.

Experiments with porous polymer monoliths according to embodiments ofthe invention have shown that analyte molecules small enough topenetrate the mesopore system have access to binding sites that arelocated inside the pore structure while the binding capacity for largeranalytes drops dramatically when mesopore access is restricted due totheir size. The result is a mixed mode retention mechanism where a sizeexclusion effect overlays the hydrophobic binding. It has been furtherdemonstrated that the binding capacity for larger analytes increaseswhen the size (eg in terms of monomer units or molecular weight) of theporogen increases while at the same time the binding capacity forsmaller analytes remains constant. This indicates that the size of theaforementioned mesostructures derived from the secondary porogens ispredictably influenced by the size of the secondary porogen oligomers.

According to a significant preferred feature of the invention, in themethod of forming a porous polymer monolith according to the secondaspect of the invention the pore size profile of the mesopores ispredetermined by the molecular size and therefore molecular weight ofthe secondary porogen oligomers.

This preferred aspect of the invention can be employed to design a rangeof monolithic materials that have a ‘tuneable’ analyte size cut-offdependent on the size of porogen employed. An exemplary application isthe pre-concentration of metabolites in serum. Blood serum contains alarge amount of proteins which, because of their size, would only have avery limited binding capacity on the monolith (being too large to accessmesopores). Metabolites on the other hand are predominantly smallmolecules with molecular weights below 500 and would have full access tothe mesoporous interior of the monolithic phase.

As already indicated, the secondary porogen is preferably soluble in thesolution containing at least a monomer, crosslinker and primary porogen.To function as required, it should predominantly remain in the solidphase during the phase separation so as to form the aforementionedmesostructures able to be washed from the polymer body.

Washing of the mesostructures from the polymer body or monolith may beeffected with any solvent suitable to dissolve and wash away themesostructures. In the aforedescribed embodiment employing styreneoligomers, a suitable such solvent would be dichloromethane.

In a preferred application, the invention further provides a samplepreparation or analytic device incorporating a porous polymer monolithaccording to the first aspect of the invention. Of particular interestis an SPE (solid phase extraction) device such as a deep well plate oran SPE cartridge, or a device for performing the Micro SPE technique ofMicro Extraction by Packed Sorbent (MEPS™).

In a further adaptation of the invention, the method of the secondaspect of the invention may be carried out in situ in such a samplepreparation or analytic device. For example, the porous polymer monolithmay be fabricated in situ in the body of the device by electromagnetic,eg. ultraviolet, initiation. For this purpose, the solution includes anappropriate radiation responsive initiator known to those skilled in theart. A suitable reagent for ultraviolet initiation is2,2-dimethoxy-2-phenylacetone (DMPA).

An advantage of the method of the invention is that the size that theporogen molecule dictates the exclusion limit for the analyte to whichthe monolith may be applied as an analyte adsorbent. By employing asmaller porogen, the monolith will now only have a high binding capacityfor small molecules while all molecules above the cut-off limit have amuch reduced chance to bind to the monolith. A suitable cut-off analytemolecular weight is in the range 500-600 because this will cover wellover 90% of all applications in pharmaceutical, environmental and foodanalyses as well as metabolomic studies.

Preferably, and in accordance with known applications of porous polymermonoliths, the macro through-pores are tuned to obtain a desired flowrate when a vacuum is applied at the device outlet or low positivepressure is applied to the device inlet.

In a further embodiment the surface of the polymer monolith is graftedwith PEGMA (Poly(Ethylene Glycol)Methyl ether methacrylate) to minimisethe impact of the hydrophobic nature of the external surface:non-specific protein adsorption from complex matrices such as humanwhole blood and plasma have been shown to impact performance. PEGMAcreates a hydrophilic external surface and minimises non-specificprotein binding adsorption on the poly(DVB) adsorbent.

In a further embodiment, the monolith is grafted to prepare a mixed-modeion exchange functionality. A strong cation exchange (SCX) functionalityhas been commonly used for the extraction of polar/non polar ionizedbase compounds from complex matrices such as human blood and plasma.Moreover, the sulfonate group gives a higher degree of hydrophilicity onthe surface to prevent non-specific protein binding.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

EXAMPLES Example 1

The different porous polymer monoliths (abbreviated in this example as“monoliths”) constituting embodiments of the first aspect of theinvention were synthesised by the process of the second aspect of theinvention, using as secondary porogens, polystyrenes with respectivemolecular weights of 906, 1300 and 1681 respectively. To determine theoptimum binding capacity and recovery of different analytes on themonoliths synthesised with the various molecular weight polystyrenes,the monoliths were probed with analytes of increasing molecular weightas follows:

Analytes Molecular weight (g/mol) 3-nitroaniline 138 peptideNH₂-GGFC-COOH 336 peptide NH₂-GFGGFG-COOH 426 peptide NH₂-GGFGGFGG-COOH541 peptide NH₂-GGFGGGGFGG-COOH 654 peptide NH₂-GGFC-COOH 768

The results for the synthesised monoliths were compared to correspondingresults obtained with commercially available DVB solid phase extractiondevices—Oasis from the Waters Corporation and Sola from Thermo FisherScientific.

FIG. 1 demonstrates the increased binding capacity for the increasingmolecular weight analytes on the various synthesised monoliths. Themonolith designated “original” was made by the standard process with nosecondary porogen. As the molecular weight of the secondary porogen wasincreased a corresponding increase in binding capacity was observed forthe increasing molecular weights of the analytes.

EXAMPLE 2

Caffeine molecular weight 194, was measured in whole human capillaryblood. A calibration curve was constructed using dilutions of 0.5 mg/mLcaffeine stock solution. The following concentrations were chosen: 3.125μg/mL, 5 μg/mL, 31.25 μg/mL and 62.5 μg/mL. Calibration analysis wascarried out by High Pressure Liquid Chromatography (HPLC) under theconditions listed below and gave a correlation of R²=0.9993 All caffeineconcentrations were calculated comparison to the calibration curveobtained.

LC Conditions Column: 250 × 4.6 mm enable C18G HPLC system: ShimadzuProminence 20A Flow rate: 1.0 ml/min Mobile phase: 16% acetonitrile with0.1% TFA Detection: 270 nm Temperature: 25° C. Sample temp: 15° C.Injection volume: 1 μL

Whole blood samples were obtained from two volunteers. The whole bloodof the first volunteer was obtained 3 hours after the volunteer hadconsumed a cup of coffee. The second volunteer had not consumed coffeefor the past 24 hours. In each case, 150 μL of blood was lysed with 1350μL of water. A solid phase extraction (SPE) procedure was conductedaccording to the following sequence of steps:

-   -   Precondition cartridge using 2 mL methanol and 2 mL water    -   Load 2 mL of samples    -   Wash with 9 mL water    -   Elute with 1 mL methanol    -   HPLC analysis

A 10 μL sample was injected for HPLC analysis.

The results are shown in FIG. 2 .

The caffeine concentration in the blood of volunteer 1 was found to be2.43 μg/mL. Volunteer 2 was correctly shown to have no measurablecaffeine. The healthy level is 1-10 μg/mL. The lethal level is 80 μg/mL.

Example 3

To demonstrate the utility of the hydrophilic surface functionality of aporous polymer monolith with PEGMA surface graft, a 0.5 mg/mL of3-nitroaniline sample was loaded onto porous polymer monolith 1681(example 1) and PEGMA grafted monolith 1681. The result showed thatsample solution stayed on top of unmodified monolith 1681 but was easilyabsorbed onto the grafted monolith 1681 without conditioning andequilibration due to the hydrophilic surface allowing the sample totransfer through the polymer.

Example 4

To demonstrate the utility of grafting of the porous polymer monolith toprepare a mixed-mode ion exchange capability,2-Acrylamido-2-methylpropane sulfonic acid (AMPS) was used as thefunctional monomer for surface grafting of porous polymer monolith 1681(example 1). In order to demonstrate the degree of grafting on themesoporous structure of monolith 1681, amitriptyline (pka=9.4, MW=277.4)was chosen as the target analyte to investigate the binding of differentAMPS-grafted 1681 monoliths.

FIG. 3 shows the increased ion exchange functionality of the increaseddegree of AMPS grafting. The amount of amitriptyline loaded was 100 μgand was completely bound on all samples. The original 1681 is purelyhydrophobic and all adsorbed amitriptyline could be eluted withmethanol. As the amount of grafted AMPS was increased the amount ofamitriptyline that was retained through ionic interactions was increasedtoo. This fraction will not desorb with methanol but requires basicelution conditions. At 3% grafting the binding capacity foramitriptyline is equally shared between the reversed phase and the ionicmode. At 10% grafting and above, the material has turned from a mixedmode sorbent to a true ion exchanger. The high recovery resultsdemonstrate a higher degree of grafting on the mesopores compared tomicropores.

1-28. (canceled)
 29. A method of making a porous polymer monolithcomprising: forming a polymer body by phase separation out of a solutioncomprising a monomer and a primary porogen, whereby the body containsmultiple macroporous through-pores, wherein the solution furthercomprises a secondary porogen comprising oligomers inert with respect tothe monomer but chemically compatible with the monomer so as to formmesostructures within the polymer body during said phase separation,wherein the secondary porogen has a molecular weight of not more than5000, and washing the mesostructures from the body to provide an arrayof mesopores such that the surface area of the monolith is predominantlyprovided by the mesopores.
 30. The method according to claim 29 whereinthe secondary porogen is an oligomeric form of the monolith polymer. 31.The method according to claim 29 wherein the secondary porogen comprisesstyrene oligomers.
 32. The method according to claim 31 wherein thestyrene oligomers are structured to minimise or prevent theirparticipation in the primary phase separation polymerisation reactionthat forms the monolith body.
 33. The method according to claim 30wherein the monomer is a mix of methacrylates and the secondary porogenis a HEMA oligomer.
 34. The method according to claim 29 wherein thereare no or substantially no residual mesostructures formed from thesecondary porogen.
 35. The method according to claim 29 wherein themacoporous through-pores are substantially unmodified and unaffected bythe addition of the secondary porogen during the formation of thepolymer body.
 36. The method according to claim 29 wherein the pore sizeprofile of the mesopores is predetermined by the molecular size andtherefore molecular weight of the secondary porogen oligomers.
 37. Themethod according to claim 29 wherein the secondary porogen is soluble inthe solution comprising the monomer and primary porogen.
 38. The methodaccording to claim 29 wherein the mesopores have a pore size in therange 20-120 Å or 40-120 Å.
 39. The method according to claim 29 whereinthe macroporous through-pores have a pore size in the range of 2-3microns or 1-5 micron.
 40. The method according to claim 29 wherein themesopores contain sites adapted to bind molecules of predeterminedcharacter, structure, chemistry or size.
 41. The method according toclaim 29 wherein greater than 65% of the surface area of the monolith isprovided by the mesopores.
 42. The method according to claim 29 carriedout in situ in a sample preparation or analytic device.
 43. The methodaccording to claim 29 further comprising surface grafting onto a surfaceof the monolith body to provide a hydrophilic external surface.
 44. Themethod according to claim 43, wherein the surface grafting comprisinggrafting poly(ethyleneglycol)methylether methacrylate (PEGMA) onto thesurface of the polymer body.
 45. The method according to claim 44wherein the monomer is divinyl benzene.
 46. The method according toclaim 29 wherein the monomer is divinyl benzene.
 47. The methodaccording to claim 29 wherein the secondary porogen has a molecularweight of not more than
 2000. 48. A method for separating an analytefrom a solution, the method comprising passing the solution comprisingthe analytye through the polymer body prepared according to the methodof claim 29.